In vivo occupation of estrogen receptors by hydroxylated metabolites of tamoxifen]

We report that after in vivo administration of (3H) tamoxifen, the cytosol and nuclear estrogen receptor sites of Rat uterus and Chicken oviduct are mostly occupied by polar metabolites. One of the major metabolites is 4-hydroxy-tamoxifen which we have identified by cocrystallisation withe non radioactive compound and which is known to display a high affinity for the estrogen receptor. In the Rat uterus, the proportion of the metabolites versus tamoxifen, increases with time with a maximum at 8 hrs. for the 4-hydroxy-tamoxifen. Other hydroxylated metabolites (M2) became predominant after 24 hrs. We propose that in vivo, the synthetic antiestrogens act mostly via their transformation into hydroxylated metabolites.     

The effect of homogenization temperature upon the apparent cellular compartmentalization of unoccupied estrogen receptor

Homogenization of rat uterus at elevated temperatures results in an increased nuclear localization of unoccupied estrogen receptor. This is a nonlinear effect which is accounted for by an increased population of KCl-resistant nuclear binding sites at the elevated homogenization temperatures.This work was supported by NSF Research Grant PCM-8409586.     

Novel estrogen receptor coregulators and signaling molecules in human diseases

The steroid hormone estrogen and signaling from its receptors are increasingly recognized as critical mediators of a variety of organ-specific biological processes. Recent advances in the identification and functional characterization of novel estrogen receptor interacting proteins clearly show the complexity of hormonal signaling regulation, but may also contribute to our understanding of the roles of estrogen signaling in normal physiology and the pathobiology of human disease.Received 12 June 2003; received after revision 21 July 2003; accepted 29 July 2003     

Affinity of 1,2-substituted oxytocin analogues to the uterus receptor: Free-Wilson and Hansch analysis.

The analysis of pA2 values for 1,2-substituted oxytocin analogues suggests a significant resonance effect of p-substituted groups in 2-tyrosine when the hormone binds to its uterus receptor, whereas the N-terminal amino group exerts less clearly characterized effects (participation of its lipophilicity and molecular volume can be assumed.     

Affinity of 1,2-substituted oxytocin analogues to the uterus receptor: Free-Wilson and Hansch analysis

Summary The analysis of pA₂ values for 1,2-substituted oxytocin analogues suggests a significant resonance effect of psubstituted groups in 2-tyrosine when the hormone binds to its uterus receptor, whereas the N-terminal amino group exerts less clearly characterized effects (participation of its lipophilicity and molecular volume can be assumed).Supported by the Swiss National Science Foundation, grant No. 3.040.76.     

17 Beta-estradiol-sensitivity of cultured myometrial cells

The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17 beta-estradiol. Following a time lag of approximately 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in vitro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.     

Uterine glucose metabolism in the prepubertal rat treated neonatally with androgen, estrogen, and antihormones

Estrogen secretion during infancy may selectively enhance the phosphogluconate oxidative pathway in the rat uterus, for altered estrogen-stimulated glucose oxidation prepubertally is correlated (+0.91) with impaired ovarian development and not uterine estrogen receptor content.     

Uterine glucose metabolism in the prepubertal rat treated neonatally with androgen,estrogen, and antihormones

Summary Estrogen secretion during infancy may selectively enhance the phosphogluconate oxidative pathway in the rat uterus, for altered estrogen-stimulated glucose oxidation prepubertally is correlated (+0.91) with impaired ovarian development and not uterine estrogen receptor content.This work was supported in part by NSF Research Grant PCM-8409586.     

Pharmacological versus binding analysis of receptor systems: how do they interplay? Myometrial cell receptors for oxytocin as a paradigm.

Binding studies in various biological systems frequently indicate the presence of several binding sites for a biologically active ligand. They differ in their affinity for the ligand in question, binding capacity, and Hill coefficient, which suggests differences in the mechanisms of the binding site-ligand interactions. Identification of the 'true' receptors (sites initiating a cellular response) appears to be difficult. Three clusters of binding sites for oxytocin were found on rat myometrial cells. The oxytocin receptor seems to be linked to the medium-affinity site; the cooperation between the high- and medium-affinity sites in eliciting the uterotonic response seems likely, but lacks experimental proof. Dose-response analysis in partially irreversibly inhibited uterus preparations, the method of equipotent doses (Furchgott-Bursztyn method), and structure-activity analysis of oxytocin-like peptides acting as competitive inhibitors of oxytocin, turned out to be suitable for pharmacological analysis of this receptor system.     

17β-Estradiol-sensitivity of cultured myometrial cells

Summary The estrogen sensitivity of cells cultured from the rat myometrium was studied by growing the cells in the absence or presence of 1 nM 17-estradiol. Following a time lag of 10 days, exposure to estrogen resulted in increased incorporation of radiothymidine by the cells. Estrogen treatment also decreased isoproterenol-dependent and GTP-dependent adenylate cyclase activity, but had no effect on basal activity. These cultured cells have been shown previously to have some properties of uterine smooth muscle. The effects estrogen has in viyro, therefore, may reflect important properties in vivo that account for the mechanism by which the sex steroid decreases the sensitivity of the myometrium to isoproterenol.     

Ethanol and opioid receptor signalling

Summary Ethanol may modulate endogenous opioid systems by disrupting opioid receptor signalling. Low concentrations of ethanol slightly potentiate -opioid receptor binding by increasing receptor B_max, and, in some cases, chronic ethanol exposure decreases the density or affinity of the -opioid receptors. By contrast, high concentrations of ethanol acutely decrease -opioid receptor binding by decreasing receptor affinity, whereas chronic exposure of animals and neuronal cell lines to lower concentrations of ethanol leads to possibly adaptive increases in the density or affinity of the -opioid receptors. In the neuronal cell line NG108-15, ethanol does not up-regulate the -opioid receptor by blocking receptor degradation or endocytosis, but protein synthesis is required for this response. Up-regulation of the -opioid receptor renders ethanol-treated NG108-15 cells 3.5-fold more sensitive to opioid inhibition of adenylyl cyclase. Long-term treatment with ethanol also increases maximal opioid inhibition in NG108-15 cells, possibly by decreasing levels of G_s and its mRNA. Ethanol differentially modulates signal transduction proteins in three additional neuronal cell lines, N18TG2, N4TG1, and N1E-115. Ethanol-treated N18TG2 cells show the least up-regulation of the -opioid receptor, little heterologous desensitization of adenylyl cyclase, and no changes in G_s or G_i. By contrast, ethanol-treated N1E-115 cells show the largest up-regulation of the -opioid receptor, the most heterologous desensitization of adenylyl cyclase, and concentration-dependent decreases in G_s and increases in G_i. Further analysis of these related neuronal cell lines may help to identify the molecular elements that endow some, but not all, neuronal cells with the capacity to adapt to ethanol.     

The enteric neural receptor for 5-hydroxytryptamine

An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using 3H-5-HT as a radioligand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of 3H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these 3H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of 3H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of 3H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of 3H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Fatty acid modulation of antiestrogen action and antiestrogen-binding protein in cultured lymphoid cells

Nonsteroidal antiestrogens reversibly and specifically inhibited the proliferation of two estrogen receptor-negative lymphoid cell lines (EL4 and Raji) in a dose-dependent manner. [3H]Thymidine incorporation of concanavalin A-stimulated primary splenocytes was also inhibited by 10(-6) M clomiphene (1-[4-(2-diethylaminoethoxy)phenyl]-1,2-diphenyl-2-chloroethylene). The antiproliferative effect could be prevented by the simultaneous presence in the growth medium of 10(-5) M linoleic acid or 10(-5) M arachidonic acid but not by 10(-6) M estradiol. Both lymphoid cell lines had high affinity antiestrogen-binding sites whose affinity could be altered by conditions of growth. Growth of EL4 cells in RPMI 1640 medium supplemented with charcoal-pretreated 5% fetal calf serum (charcoal-stripped medium) resulted in significantly higher affinity (Kd 0.54 nM +/- 0.11 nM; n = 6) than growth in medium supplemented with untreated serum (complete medium) (Kd = 1.68 nM +/- 0.48 nM; n = 6) (p less than 0.001). This change in affinity was partly due to removal of fatty acids from the growth medium by charcoal pretreatment, since addition of 10(-5) M linoleic acid or 10(-5) M gamma-linolenic to charcoal-stripped medium decreased the affinity of the antiestrogen-binding protein. In contrast, growth in 10(-5) M stearic acid or 10(-5) M oleic acid did not significantly alter the affinity of the antiestrogen-binding protein, whereas 10(-5) M palmitic acid significantly increased its affinity. The same fatty acids were also tested for their intrinsic effects on EL4 cell proliferation. Oleic, linoleic and gamma-linolenic acids were growth stimulatory while stearic and palmitic acids were not. Thus linoleic and gamma-linolenic acids whose presence in the growth medium was associated with decreased affinity of [3H]tamoxifen (1-[4-(2-dimethylaminoethoxy)phenyl]-1,2-diphenylbut-1(Z)-ene) binding to the intracellular antiestrogen-binding protein were also growth stimulatory. Unsaturated fatty acids have previously been shown to inhibit binding of [3H]tamoxifen to the antiestrogen-binding protein in a cell-free system. The present observations demonstrate that unsaturated fatty acids also modify the affinity of the antiestrogen-binding protein in intact cells.     

Pharmacological versus binding analysis of receptor systems: How do they interplay? Myometrial cell receptors for oxytocin as a paradigm

Summary Binding studies in various biological systems frequently indicate the presence of several binding sites for a biologically active ligand. They differ in their affinity for the ligand in question, binding capacity, and Hill coefficient, which suggests differences in the mechanisms of the binding site-ligand interactions. Identification of the true receptors (sites initiating a cellular response) appears to be difficult. Three clusters of binding sites for oxytocin were found on rat myometrial cells. The oxytocin receptor seems to be linked to the medium-affinity site; the cooperation between the high-and medium-affinity sites in eliciting the uterotonic response seems likely, but lacks experimental proof. Dose-response analysis in partially irreversibly inhibited uterus preparations, the method of equipotent doses (Furchgott-Bursztyn method), and structure-activity analysis of oxytocin-like peptides acting as competitive inhibitors of oxytocin, turned out to be suitable for pharmacological analysis of this receptor system.     

Synthesis and cannabinoid receptor binding activity of conjugated triene anandamide,a novel eicosanoid

A polyenoic fatty-acid isomerase (PFI) from a red marine alga was used to convert anandamide (5Z,8Z,11Z,14Z-eicosatetraenoyl-N-ethanolamide) to the 5Z,7E,9E,14Z-eicosatetraenoyl-N-ethanolamide isomer. This novel eicosanoid, termed conjugated triene anandamide (CTA), was assessed for its ability to bind to the cannabinoid receptor in rat brain membrane preparations. CTA is a high affinity cannabimimetic substance whose novel structure provides new insight into structure-activity relationships of cannabinoid receptor ligands. These experiments illustrate the utility of enzymes isolated from marine organisms in the development of pharmacological probes.     

Evidence for differences in alpha-adrenergic receptor affinity in stress susceptible swine

Summary pA₂-Values were determined using phentolamine-methoxamine. The mean pA₂-value on aortic strips from stress susceptible swine was 7.81 and 7.39 for control. The -adrenergic receptor from stress susceptible swine has a higher affinity than that of control.Acknowledgments. The author appreciates the cooperation of Dr L. L. Christian and the Meat Laboratory in obtaining the tissues and the technical assistance of Vern Hoyt.     

The enteric neural receptor for 5-hydroxytryptamine

Summary An enteric neural receptor for serotonin (5-HT) has been characterized. This receptor was assayed, using³H-5-HT as a radiologand, by rapid filtration of isolated enteric membranes and by radioautography. In addition, intracellular recordings were made from ganglion cells of the myenteric plexus. High affinity, saturable, reversible, and specific binding of³H-5-HT was demonstrated both to membranes of the dissected longitudinal muscle with adherent myenteric plexus and the mucosa-submucosa. Radioautographs showed these³H-5-HT binding sites to be in myenteric ganglia and in a broad unresolved band at the mucosal-submucosal interface. Antagonists active at receptors for other neurotransmitters than 5-HT, at either of the two known types of CNS 5-HT receptor, and at 5-HT uptake sites on serotonergic neurons failed to inhibit binding of³H-5-HT. The structural requirements of analogues for binding to the enteric 5-HT receptor matched the known pharmacology of M or neural 5-HT receptors. A novel 5-HT antagonist was found. This compound, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide (5-HTP-DP), antagonized the action of 5-HT on type II/AH cells of the myenteric plexus but did not affect the release or actions of acetylcholine (nicotinic or muscarinic) or substance P. 5-HTP-DP was also an equally potent displacer of³H-5-HT from its binding sites on enteric membranes. It is concluded that the sites responsible for specific binding of³H-5-HT are enteric M or neural 5-HT receptors. These receptors differ from those now known to be present in the CNS.     

Characterization of the "4 S-trypsin" form of the cytosoluble estradiol receptor of calf uterus in a nondenaturing system]

The characterisation of the "4 S-trypsin" form of the estradiol-receptor from calf uterus cytosol was carried out by polyacrylamide gel electrophoresis under non-denaturing conditions. In a multiphasic buffer system (upper buffer Tris-glycine pH25degrees C = 8,6, lower buffer Tris HCl pH25degrees C = 7,4), three radioactive estradiol peaks were observed. The first was free estradiol, the second estradiol initially complexed with a macromolecule and dissociated during electrophoresis, and the third the hormone-receptor complex. The sedimentation coefficient (4 S) of this complex was the same before and after electrophoresis. Its mean geometric radius is R = 2.53 + 0.08 nm and its molecular weight was estimated to be 55,000.     

Binding of estradiol to synaptosomal mitochondria: physiological significance

The subsynaptosomal distribution and specific binding of 17beta-estradiol in vitro to mitochondria isolated from presynaptic nerve endings of female rat brain were examined. 17Beta-estradiol is (i) distributed unequally in synaptosomes and mitochondria posses the highest capacity to bind estradiol with respect to the available amount of the hormone. (ii) Estradiol binds specifically to isolated synaptosomal mitochondria. A Michaelis-Menten plot of specific binding was sigmoidal within a concentration range of 0.1-5 nM of added estradiol, with a saturation plateau at 3 nM. Binding of higher estradiol concentrations demonstrated an exponential Michaelis-Menten plot, indicating non-specific binding to mitochondria. Vmax and Km for the sigmoidal-shape range were estimated as 46 +/- 6 fmol of estradiol/mg of mitochondrial proteins and 0.46 +/- 0.07 nM free estradiol respectively. (iii) Estradiol binding is not affected by the removal of ovaries. The results show that inhibition of Na-dependent Ca2+ efflux from mitochondria by estradiol occurs according to an affinity change of the translocator for Na+, at the same estradiol concentrations that show specific binding to mitochondrial membranes. These data imply that physiological concentrations of estradiol, acting on mitochondrial membrane properties, extragenomically modulate the mitochondrial, and consequently the synaptosomal content of Ca2+, and in that way exert a significant change in nerve cell homeostasis.